Interpretive Summary: Outbreaks of hepatitis A virus and human norovirus pose significant global public health challenges. Diagnostic testing for viral contaminants is imperative to ensure food safety, especially for consumers of raw shellfish, which are frequently associated with virus outbreaks. Current tissue-based shellfish testing methods are tedious, time-consuming, and are not practical for routine monitoring. Rapid, simple, and cost-effective methods are needed to detect viruses in shellfish. The goal of the experiments described here was to determine whether oyster blood cells called hemocytes could be utilized in rapid viral extraction procedures, and to compare them to a tissue-based method that uses whole oysters. In this study, virus-contamination was detected in oysters with two hemocyte RNA extraction methods which were evaluated in a fraction of the time and with improved sensitivity as compared to the whole tissue-based protocol. These results support the finding that oyster hemocytes may be a source for rapid detection of viral pathogens in oysters, thus improving upon current time-consuming methods using whole or dissected tissues. These innovative hemocyte-based methods could be applied to routine monitoring of shellfish to prevent foodborne illness and to ensure safe shellfish for consumers.

Technical Abstract:
Rapid viral detection methods are necessary to employ diagnostic testing for viral contamination in shellfish to prevent and control foodborne illness. Current shellfish viral RNA extraction methods, which are time-consuming and not applicable for routine monitoring, require the testing of whole or dissected tissues. Our laboratory previously showed that Eastern oyster (Crassostrea virginica) hemocytes are a site of viral persistence. Therefore, hemocytes might be utilized in screening for contaminated shellfish instead of processing oyster tissues. In this study, a duplex qRT-PCR assay was used to compare the detection of hepatitis A virus and murine norovirus-contaminated oysters in RNA extracts prepared from hemocytes and whole oysters. RNA extracts were prepared from oyster hemocytes using Invitrogen Dynabeads Oligo(dT)(25) and the Qiagen RNeasy Mini Kit, and were compared to RNA extracts prepared with a tissue-based laboratory method using whole oysters. Virus-contamination was detected with higher sensitivity in oysters using both hemocyte RNA extraction methods and extraction was performed in a fraction of the time required for the tissue-based RNA extraction method using whole oysters. These data suggest that the analysis of oyster hemocytes may be a rapid and useful tool to monitor shellfish safety.